Claims:

1. A method of promoting protection or repair of articular cartilage in a
subject comprising a. selecting a subject in need of protection or repair
of articular cartilage; and b. administering intermittently to the
subject a parathyroid hormone/parathyroid hormone-related protein
(PTH/PTHrP) receptor agonist.

2. The method of claim 1, wherein the subject in need of protection or
repair of articular cartilage has injured articular cartilage.

3. The method of claim 1, wherein the articular cartilage is injured by
trauma.

4. The method of claim 1, wherein the articular cartilage is injured by
or treated by surgery.

5. The method of claim 4, wherein the surgery is selected from the group
consisting of arthroscopic shaving of cartilage, chondroplasty, and
mosaicplasty.

7. The method of claim 4, wherein the surgery comprises implantation of a
scaffolding material or composite.

8. The method of claim 1, wherein the articular cartilage is injured by
an ischemic event.

9. The method of claim 1, wherein the articular cartilage is injured by a
disease or condition.

10. The method of claim 9, wherein the disease or condition is an
infection.

11. The method of claim 9, wherein the disease or condition is inherited.

12. The method of claim 9, wherein the disease or condition is an
ischemic condition.

13. A method of promoting protection or repair of musculoskeletal soft
tissue in a subject comprising a. selecting a subject in need of repair
of musculoskeletal soft tissue; and b. administering intermittently to
the subject a parathyroid hormone/parathyroid hormone-related protein
(PTH/PTHrP) receptor agonist.

14. The method of claim 13, wherein the musculoskeletal soft tissue is
cartilage.

15. The method of claim 14, wherein the cartilage is a meniscal
cartilage.

16. The method of claim 13, wherein the musculoskeletal soft tissue is a
ligament.

17. The method of claim 13, wherein the musculoskeletal soft tissue is a
tendon.

18. The method of claim 13, wherein the musculoskeletal soft tissue is a
skeletal muscle.

19. The method of claim 13, wherein the musculoskeletal soft tissue is an
intervertebral disc.

20. The method of claim 13, wherein the subject in need of repair of
musculoskeletal soft tissue has injured musculoskeletal soft tissue.

21. The method of claim 20, wherein the musculoskeletal soft tissue is
injured by trauma.

22. The method of claim 20, wherein the musculoskeletal soft tissue is
injured by or treated by surgery.

24. The method of claim 22, wherein the surgery comprises implantation of
a scaffolding material or composite.

25. The method of claim 20, wherein the musculoskeletal soft tissue is
injured by an ischemic event.

26. The method of claim 20, wherein the musculoskeletal soft tissue is
injured by a disease or condition.

27. The method of claim 26, wherein the disease or condition is an
infection.

28. The method of claim 26, wherein the disease or condition is
inherited.

29. The method of claim 26, wherein the disease or condition is an
ischemic condition.

30. The method of claim 1, wherein the PTH/PTHrP receptor agonist is a
peptide agonist.

31. The method of claim 30, wherein the peptide agonist is teriparatide.

32. The method of claim 1, wherein subject does not have osteoporosis.

33. The method of claim 1, wherein subject does not have a bone fracture.

34. A method of protecting or promoting repair of articular cartilage in
a subject comprising a. selecting a subject in need of protection or
repair of articular cartilage; and b. administering intermittently to the
subject a parathyroid hormone releasing factor.

35. A method of promoting repair of musculoskeletal soft tissue in a
subject comprising a. selecting a subject in need of repair of
musculoskeletal soft tissue; and b. administering intermittently to the
subject a parathyroid hormone releasing factor.

Description:

[0003] There is currently no known medical treatment for stimulating the
protection or repair of cartilage, tendons, muscles, meniscus,
intervertebral discs, or ligaments. Osteoarthritis, for example, which is
associated with degeneration of articular cartilage, is a leading cause
of disability and immobility, with current estimates of over 40 million
Americans affected. Articular cartilage in adults has an extremely
limited ability to repair itself, and once deterioration begins, the
outcome is usually an irreversible progressive degeneration and
associated inflammation, pain and dysfunction. Current treatments include
anti-inflammatory and pain medications, injections of steroids and
hyaluronic acid into the affected joint, and surgeries, such as total
joint replacement. Oral supplements of glucosamine and chondroitin
sulfate have been proposed as chondroprotective, but the
chondroprotective effects of such oral supplements are controversial and
minimal at best.

SUMMARY

[0004] Provided herein are in vivo and in vitro methods of promoting
protection or repair of articular cartilage and/or musculoskeletal soft
tissue (e.g., non-articular cartilage like meniscal cartilage,
intervertebral disc, skeletal muscle, tendon, or ligament) using a
parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP)
receptor agonist or releasing agent, For example, provided herein is a
method of promoting protection or repair of articular cartilage and/or
musculoskeletal soft tissue in a subject comprising selecting a subject
in need of such protection or repair and administering intermittently to
the subject a PTH/PTHrP receptor agonist. Also provided is a method of
protecting or promoting repair of articular cartilage and/or
musculoskeletal soft tissue in a subject comprising selecting a subject
in need of protection or repair of articular cartilage and administering
intermittently to the subject a parathyroid hormone releasing factor.

[0007] FIG. 2B shows two micrographs, one from a normal human articular
cartilage (NL) and one from an osteoarthritic human articular cartilage
(OA), showing immunohistochemical labeling using an antibody to the
parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP)
receptor.

[0008] FIG. 3A is a histogram showing expression of p3tp-luciferase levels
relative to control (i.e., untreated) in chick sternal chondrocytes
cultured with transforming growth factor beta (TGFβ) and transiently
transfected with smurf2, alone or in combination, or treated with
TGFβ and parathyroid hormone-related peptide (PTHrP) with a chronic
exposure for 48 hours.

[0011] FIG. 3D shows images of Western blots demonstrating that the
reduction of pSmad2 and pSmad3 by PTHrP is reversed by inhibiting Smurf2
expression with siRNAs. The Western blots show pSmad3 and pSmad2 levels
in chick sternal chondrocytes cultured in the presence or absence of
TGFI3, PTHrP and/or a Smurf2 RNAi (Sf2 RNAi) for 48 hours.

[0012] FIG. 4 shows micrographs of articular cartilage in mice with a
murine knee injury model (mild injury and severe injury) and sham mice at
4, 8, and 12 weeks following surgery and with 4, 8, or 12 weeks of daily
treatment with teriparatide (+PTH). FIG. 4A shows micrographs of mice
with a murine injury model and sham mice at 4 weeks following surgery and
with 4 weeks of daily treatment with teriparatide. FIG. 4B shows
micrographs of mice with a murine injury model and sham mice at 8 weeks
following surgery and with 8 weeks of daily treatment with teriparatide.
FIG. 4C shows micrographs of mice with a murine injury model and sham
mice at 12 weeks following surgery and with 12 weeks of daily treatment
with teriparatide.

[0013] FIG. 5 shows micrographs of normal and injured human meniscus.
Panels on the left (5A, 5C, and 5E) show normal samples labeled
immunohistochemically with an antibody to PTH/PTHrP receptor, whereas
panels on right (5B, 5D, and 5F) show injured meniscus samples. Top
panels (5A and 5B) are at 10× magnification; remaining panels
(5C-5F) are at 20× magnification. All panels show white zones of
the meniscus, except for panel 5C, which shows the red zone of a normal
meniscus.

[0014] FIG. 6 shows a histogram demonstrating that chondrocyte markers
(MMP-13 and ADAMTS5) consistent with osteoarthritis were upregulated in
mice with meniscal injury. Additionally, the histogram shows upregulation
of PTHR1 in osteoarthritic cartilage in the mice.

[0015] FIGS. 7A and 7B show that daily injection of teriparatide or PTH
immediately following sham or meniscus/ligament injury stimulated the
production of proteoglycan in the articular cartilage. FIG. 7A shows
images of micrographs demonstrating that proteoglycan production is
stimulated by treatment with teriparatide and PTH for 4 weeks following
surgery. FIG. 7B shows a histogram quantifying the Alcian Blue staining
intensity demonstrating an increase in proteoglycan production following
treatment with teriparatide and PTH for 4 weeks following surgery.

[0016] FIGS. 8A and 8B show that immediate treatment with teriparatide and
PTH after meniscus/ligament injury for 12 weeks results in a reduction in
cartilage loss. FIG. 8A shows images of micrographs demonstrating that
treatment with teriparatide and PTH for 12 weeks reduces the amount of
cartilage loss following surgery. FIG. 8B shows a histogram quantifying
the normalized cartilage area following treatment with teriparatide and
PTH for 12 weeks following surgery. Treatment with teriparatide or PTH
reduces the amount of cartilage loss as compared to treatment with
saline.

[0017] FIGS. 9A and 9B show that delayed treatment with teriparatide and
PTH 8 weeks after meniscus/ligament injury results in a reduction in
cartilage loss. FIG. 9A shows images of micrographs demonstrating that
treatment with teriparatide and PTH for 4 weeks after an 8 week delay
post surgery reduces the amount of cartilage loss as compared to
treatment with saline. FIG. 9B shows a histogram quantifying the
normalized cartilage area following treatment with teriparatide and PTH
for 4 weeks after an 8 week delay post surgery. Delayed treatment with
teriparatide or PTH reduces the amount of cartilage loss as compared to
treatment with saline.

[0018]FIG. 10 shows a histogram demonstrating treatment of rabbit
mensical cells with PTH for 3 days resulted in an upregulation of cyclin
D1 mRNA, indicating a stimulation of meniscal cell proliferation.

[0019] FIG. 11 shows a histogram demonstrating treatment of rabbit
meniscal cells with PTH resulted in an upregulation of type I collagen,
the predominant matrix molecule in the meniscus, indicating a beneficial
effect of PTHR1 stimulation on meniscal healing.

[0020] FIGS. 12A and 12B show daily treatment with teriparatide for 4
weeks following meniscal surgery results in preliminary meniscal healing.
FIG. 12A shows a micrograph of a white zone tear in a rabbit meniscus
treated with saline for 4 weeks as a control. FIG. 12B shows a micrograph
of a white zone tear in a rabbit meniscus treated daily with teriparatide
for 4 weeks. Treatment with teriparatide shows narrowing of the defect
with increased cellularity and proteoglycan content consistent with a
stimulatory healing effect on the meniscus.

DETAILED DESCRIPTION

[0021] The parathyroid hormone/parathyroid hormone-related protein
(PTH/PTHrP) receptor, also known as the Type 1 PTH receptor (PTHR1), is
not expressed in normal adult articular cartilage but is expressed in
chondrocytes early in the degenerative process. PTH/PTHrP receptor
expression occurs in cartilage as part of normal endochondral bone
formation and in the cartilaginous growth plates of children. The
receptor and its ligands (PTH and PTHrP) regulate endochondral
ossification, in which cartilage mineralizes and forms bone. PTHrP
stimulates growth plate chondrocyte proliferation and proteoglycan
synthesis. In addition to stimulating cell proliferation and matrix
synthesis by growth plate chondrocytes, PTH and PTHrP inhibit the
subsequent events of the endochondral ossification cellular program,
which include cellular hypertrophy, expression of matrix-degrading
enzymes (matrix metalloproteinases), expression of a type of collagen
(Type X collagen) associated very specifically with cartilage
mineralization, and ultimately, apoptosis of the chondrocytes to make way
for bone cells to convert the mineralized cartilage into bone. These
phenomena characterize the hypertrophic phenotype of chondrocytes, which
represents the terminal chondrocyte maturational program resulting in
mineralization and cellular apoptosis. PTHrP expression in the growth
plate is in turn regulated by another factor, Indian Hedgehog (IHH). This
program controls chondrocyte proliferation and hypertrophy at the growth
plate but activation of this pathway in articular cartilage is a
contributing factor in the pathophysiology of articular cartilage
degeneration.

[0022] Regulatory factors known to be expressed in the growth plate (e.g.,
PTHrP, IHH, PTH/PTHrP receptor, matrix metalloproteinase 9 (MMP9), matrix
metalloproteinase 13 (MMP13), and bone morphogenetic protein 6 (BMP6))
are absent in normal human adult articular cartilage but are expressed
early in cartilage degeneration. Furthermore, PTHrP in osteoarthritic
cartilage is expressed in groups of cloning (i.e., proliferating)
chondrocytes. Enhanced proteoglycan synthesis also occurs early after
cartilage injury, and is also thought to represent a cellular attempt at
tissue repair. However, almost invariably, these cellular activities are
insufficient to repair the tissue or halt progressive cartilage
degeneration. Similar events are observed in various types of cartilage
(e.g., articular and meniscal) and other musculoskeletal soft tissues,
e.g., skeletal muscle tissue, intervertebral discs, ligaments, and
tendons. The methods herein are designed to allow repair and to prevent
degeneration of articular cartilage, skeletal muscle, non-articular
cartilage (including meniscal cartilage), ligaments, tendons, and
intervertebral discs.

[0023] The reparative actions of intermittent activation of the PTH/PTHrP
receptor in skeletal tissues include stimulation of matrix synthesis,
usually collagen and proteoglycans, and cellular proliferation.
Mesenchymal stem cells also respond to PTH/PTHrP receptor stimulation and
are activated at and/or recruited to sites of musculoskeletal injury to
enhance repair of tissues.

[0024] The methods include the steps of selecting a subject in need of
protection or repair of the selected tissue and administering
intermittently to the subject a PTH/PTHrP receptor agonist or a
parathyroid releasing factor or stimulus. Intermittent stimulation of the
PTH/PTHrP receptor stimulates the mesenchymally derived stem cells in the
target tissue but, unlike continuous administration, does not facilitate
escape of cells like chondrocytes from the proliferative stage to the
hypertrophic state.

[0025] The subject in need of cartilage (e.g., articular or meniscal
cartilage) protection or repair may have injured cartilage or may be at
risk for cartilage degeneration. Cartilage injuries include, but are not
limited to, traumatic injury (e.g., an internal derangement, a fracture
of an articular surface, blunt injury) and surgical injury (e.g.,
associated with arthroscopic shaving or abrasion of cartilage,
chondroplasty, drilling, meniscus repair, or mosaicplasty). The subject
in need of cartilage protection or repair may have a disease or condition
or may be at risk for such disease or condition, such as osteoarthritis,
infection (e.g., a joint infection), deformity (resulting, e.g., in
abnormal joint load), gout, and calcium pyrophosphate deposition disease.
Such injuries, diseases or conditions may be associated with a defect in
a cartilage or articular surface or may put the subject at risk for
degradation of cartilage. For example, one of skill in the art could
select a subject in need of cartilage protection or repair based on
epidemiological data that the injury, condition or disease is known to be
associated with an increased risk of osteoarthritis or may use imaging
techniques or other clinical tests to detect a defect in the cartilage.
Clinical signs, for example, could include swelling, pain, and joint
crepitance or instability.

[0026] The PTH/PTHrP receptor agonist is optionally a peptide agonist or a
small molecule. Examples of receptor agonists include N-terminal peptides
or peptide analogs of PTH or PTHrP. One example of an N-terminal peptide
is teriparatide) (FORTEO®) (Eli Lilly and Company; Indianapolis,
Ind.).

[0027] Also useful in the methods taught herein are agents that stimulate
release of PTH. By way of example, calcium receptor blocking drugs
function to disrupt normal signaling in the parathyroid glands and to
induce a burst of PTH release from the glands. The released PTH,
stimulates the PTH/PTHrP receptor as a PTH/PTHrP agonist. The PTH/PTHrP
releasing factor is optionally a peptide agonist or small molecule (e.g.,
ronacaleret, manufactured by GlaxoSmithKline; Philadelphia, Pa.).

[0028] Similar methods are useful in the protection and repair of
musculoskeletal soft tissues such as non-articular cartilage, ligament,
tendon, skeletal muscle (including, e.g., sphincter muscles like the
pelvic floor and the urethral sphincter muscle), meniscus, or
intervertebral disc. In these tissues, intermittent stimulation of
PTH/PTHrP receptor expressing cells can be expected to enhance matrix
synthesis, cell proliferation, and activation or recruitment of
reparative mesenchymal stem cell. Thus, provided herein is a method for
protecting or repairing skeletal muscle, ligament, tendon or
interverterbral disc in a subject. Such a method includes the steps of
selecting a subject in need of protection or repair of a skeletal muscle,
tendon, ligament, or intervertebral disc and administering intermittently
to the subject a parathyroid hormone/parathyroid hormone-related protein
(PTH/PTHrP) receptor agonist.

[0029] As described above with regard to selection of a subject in need of
cartilage repair or protection, one of skill in the art can select a
subject in need of protection or repair of a muscle, ligament, tendon or
interverterbral disc based on epidemiological data, imaging techniques,
or clinical signs and symptoms. For example, an intervertebral disc may
be herniated or compressed because of surgery, injury, spinal deformity,
or arthritis. Disc herniation or compression can be associated with pain
and nerve impingement. Furthermore, an injury to the spine (e.g., an
athletic injury or automobile injury) could put a subject at risk of
developing a degenerative disc disease or condition. Spinal surgery or
surgical repair of a herniated disc may also mean a subject is in need of
intervertebral disc repair or protection according to the present
methods.

[0030] Similarly, an injury or surgery could indicate a subject is in need
of protection or repair of a tendon or ligament. Stretching or tearing of
a tendon or ligament, as evidenced by joint laxity, pain, clinical signs,
imaging, or epidemiological data. A subject may be in need of tendon or
ligament protection, or stimulation of repair, following trauma to or
surgical repair of a tendon or ligament.

[0032] Optionally, in the methods taught herein, the subject does not have
osteoporosis or a bone fracture. However, the methods herein can be
combined with various other treatments. For example, the methods can be
combined with chondrocyte or mesenchymal stem cell implantation to a
joint, or a traumatic or surgically created defect in the articular
surface with or without a scaffolding material to retain the implanted
cells in place. As another example, chondrocytes or mesenchymal stem
cells capable of differentiating into chondrocytes can be implanted into
a degenerating intervertebral disc of a subject and the PTH/PTHrP agonist
administered intermittently to promote repair by the implanted
chondrocytes. Similarly, a tendon or ligament graft can be accompanied by
administration of a PTH/PTHrP agonists.

[0033] For purposes of the methods taught herein, intermittent
administration of the PTH/PTHrP receptor agonist or PTH releasing factor
can be performed 1-2 times daily or every two, three, four, five, six or
seven days. In some cases, one administration may be sufficient for
preventative purposes; but generally, one of skill in the art will
determine that multiple treatments are necessary. In certain cases, local
administration may be used. For example, when a subject is undergoing
surgery, the PTH/PTHrP receptor agonist may be injected into the joint or
site of repair. Generally, however, systemic injection will be used to
avoid side effects such as infection or swelling.

[0034] The agonist or releasing factor can be administered by any route,
including oral, rectal, sublingual, ocular, and parenteral
administration. Parenteral administration includes, for example,
intrathecal, intravenous, intramuscular, intra-arterial, intraperitoneal,
intranasal, intravaginal, intraocular, intravesical (e.g., to the
bladder), intradermal, transdermal, topical or subcutaneous
administration. Also contemplated is the instillation of a drug in the
body of the subject in a controlled formulation, with systemic or local
release of the drug to occur at a later time. For example, the drug may
be localized in a depot at the site of intervention for subsequent
release to a local site, e.g., at the site of damage or surgical
intervention. Advantageously, the agents are administered in the form of
a pharmaceutical composition.

[0035] One or more agonists or releasing factors useful in the practice of
the methods described herein may be administered simultaneously, by the
same or different routes, or at different times during treatment. The
compounds may be administered before, along with, or after other
medications, including other compounds. For example, the agonists or
releasing factors could be administered with anti-inflammatory agents.

[0036] The treatment using methods described herein may be carried out for
as long a period as necessary, either in a single, uninterrupted session,
or in discrete sessions. The treating physician will know how to
increase, decrease, or interrupt treatment based on subject's response.
Optionally, treatment is carried out 1, 2, 3, 4 weeks; 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12 months; 2 years or more. One of skill in the art can
monitor the subject for any adverse side-effects, including development
of osteosarcoma and for improvement in clinical status and adjust the
treatment accordingly. The treatment schedule may be repeated as
required.

[0037] Also provided herein is a scaffold composite or material comprising
mesenchymal stem cells and a PTH/PTHrP receptor agonist (e.g., a peptide
agonist or a small molecule as described herein) or a PTH/PTHrP releasing
factor (e.g., a peptide agonist or small molecule described herein). Also
provided is a scaffold composite or material comprising mesenchymal stem
cells treated with a PTH/PTHrP receptor agonists or a PTH/PTHrP releasing
factor prior to addition of the cells to the scaffold material or
composite. Such scaffolding materials include, for example, a
biodegradable scaffolding material designed for insertion into a
cartilage defect or defect in a skeletal muscle, intervertebral disc,
ligament, or tendon. Examples of scaffold composites and materials and
methods of making and using them can be found for example in Abe et al.,
Tissue Engineering, 10: 585-594 (2004); Orthopedic Tissue Engineering
Basic Science and Practices, by Goldberg et al.; published by Informa
Health Care (2004); U.S. Pat. Nos. 7,208,177; 6,737,073; 6,530,956;
6,179,871; 5,842,477; 5,624,463; US Patent Application Nos. 20080195211;
20060273279; 20060111778; 20020009477.

[0038] Also provided is a method of treating a defect in articular
cartilage or musculoskeletal soft tissue (e.g., non-articular cartilage,
tendon, ligament, intervertebral disc, or skeletal muscle) in a subject
including the steps of inserting into the defect chondrocytes or
mesenchymal stem cells and/or a woven or unwoven scaffold material or
composite described herein. The scaffold material or composite optionally
includes mesenchymal stem cells, chondrocytes, myocytes, fibroblasts, or
a combination thereof. Optionally the cells in the scaffold are
pretreated with a PTH/PTHrP receptor agonist or a PTH/PTHrP releasing
factor. Optionally, the scaffold material contains or is impregnated with
a PTH/PTHrP receptor agonist or releasing agent. Alternatively or in
addition, the method further comprises either administering to the
scaffold or to the subject a PTH/PTHrP receptor agonist or a PTH/PTHrP
releasing factor. Optionally, the scaffold contains the PTH/PTHrP
receptor agonist or a PTH/PTHrP releasing factor prior to insertion into
the defect. Optionally, the PTH/PTHrP receptor agonist or a PTH/PTHrP
releasing factor is administered to the subject after insertion of the
scaffold, and such administration can be local or systemic and such
administration is repeated intermittently as described herein.

[0039] Provided herein are uses of the agonist or releasing agent for
preparing a medicament for protecting or repairing articular cartilage
and musculoskeletal soft tissues such as non-articular cartilage, tendon,
ligament, meniscus, intervertebral discs, and skeletal muscle. Further
provided is a use of the agonists or releasing factor for protecting or
repairing articular cartilage and musculoskeletal soft tissues such as
non-articular cartilage, tendon, ligament, intervertebral discs, and
skeletal muscle. The compounds may be administered in the form of a
pharmaceutical composition, in combination with a pharmaceutically
acceptable carrier. The amount of compound in such formulations may
comprise from 0.1 to 99.99 weight percent of the composition.
Pharmaceutically acceptable carrier means any carrier, diluent or
excipient which is compatible with the other ingredients of the
formulation and not deleterious to the recipient.

[0040] The agonist or releasing factor may be administered with a
pharmaceutically acceptable carrier selected on the basis of the selected
route of administration and standard pharmaceutical practice. The
compound may be formulated into dosage forms according to standard
practices in the field of pharmaceutical preparations. See Remington: The
Science and Practice of Pharmacy, 21st Edition, Lippincott Williams
& Wilkins, Philadelphia, Pa. (2005). Suitable dosage forms may comprise,
for example, tablets, capsules, solutions, parenteral solutions, troches,
suppositories, or suspensions.

[0041] For parenteral administration, the compound can be mixed with a
suitable carrier or diluent. Various diluents are known such as water,
oil (particularly a vegetable oil), ethanol, saline solution, aqueous
dextrose (glucose) and related sugar solutions, glycerol, or a glycol
such as propylene glycol or polyethylene glycol. One of skill in the art
would select a carrier that avoids degradation of the active agent. For
example, the carrier can be a water soluble carrier, including, for
example, an albumen solution. Solutions for parenteral administration can
contain a water soluble salt of the compound. Stabilizing agents,
antioxidant agents and preservatives may also be added. Suitable
antioxidant agents include sulfite, ascorbic acid, citric acid and its
salts, and sodium EDTA. Suitable preservatives include benzalkonium
chloride, methyl- or propyl-paraben, and chlorbutanol. The composition
for parenteral administration can take the form of an aqueous solution,
dispersion, suspension, or emulsion.

[0042] For oral administration, the compound can be combined with one or
more solid inactive ingredients for the preparation of tablets, capsules,
pills, powders, granules or other suitable oral dosage forms. For
example, the active agent can be combined with at least one excipient
such as fillers, binders, humectants, disintegrating agents, solution
retarders, absorption accelerators, wetting agents, absorbents, or
lubricating agents. According to one tablet embodiment, the active agent
can be combined with carboxymethylcellulose calcium, magnesium stearate,
mannitol, and starch, and then formed into tablets by conventional
tableting methods.

[0043] The specific dose of the agonist or releasing agent required to
obtain therapeutic benefit in the methods of treatment described herein
will, of course, be determined by the particular circumstances of the
individual patient including the size, weight, age and sex of the
patient, the nature and stage of the condition being treated, and the
route of administration of the compound. For example, a daily dosage of
teriparatide is generally about 20 μg daily for an adult human, using
a subcutaneous injection. Higher or lower doses and alternative means of
administration are also contemplated.

[0044] The agent may be formulated in a unit dosage form. The term unit
dosage form refers to physically discrete units suitable as a unitary
dosage for human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable pharmaceutical
excipient.

[0045] The pharmaceutical compositions described herein may also be
formulated so as to provide slow or controlled release of the compound
therein using, for example, hydroxypropylmethyl cellulose in varying
proportions to provide the desired release profile, other polymer
matrices, gels, permeable membranes, osmotic systems, multilayer
coatings, microparticles, liposomes and/or microspheres.

[0046] In general, a controlled-release preparation is a pharmaceutical
composition capable of releasing the compound at the required rate to
maintain constant pharmacological activity for a desirable period of
time. Since the receptor stimulation must be intermittent, however, the
controlled-release of the compound may be stimulated by various inducers,
for example pH, temperature, enzymes, water, or other physiological
conditions or compounds so that the release is intermittent.

[0047] The components used to formulate the pharmaceutical compositions
are of high purity and are substantially free of potentially harmful
contaminants (e.g., at least National Food grade, generally at least
analytical grade, and more typically at least pharmaceutical grade).
Particularly for human consumption, the composition is preferably
manufactured or formulated under Good Manufacturing Practice standards as
defined in the applicable regulations of the U.S. Food and Drug
Administration. For example, suitable formulations may be sterile and/or
substantially isotonic and/or in full compliance with all Good
Manufacturing Practice regulations of the U.S. Food and Drug
Administration.

[0048] Peptides for use in the pharmaceuticals can be made using various
techniques known to one of skill in the art. The term peptide or
polypeptide are used interchangeably herein and refers to two or more
amino acids linked by a peptide bond. The peptides can be isolated from
tissue, cells, or cultures. They can be made by proteolytically cleaving
a larger polypeptide or protein, using recombinant technology, or by
peptide synthesis methods. By way of example, one of skill in the art can
modify and design peptide agonists starting from the known sequences for
PTH and PTHrP. The sequence for human parathyroid hormone is MIPAKDMAKV
MIVMLAICFL TKSDGKSVKK RSVSEIQLMH NLGKHLNSME RVEWLRKKLQ DVHNFVALGA
PLAPRDAGSQ RPRKKEDNVL VESHEKSLGE ADKADVNVLT KAKSQ (SEQ ID NO:1) (the
first 34 amino acid residues know to bind the PTH/PTHrP receptor are
underlined). The sequence for parathyroid hormone-related peptide is
MQRRLVQQWS VAVFLLSYAV PSCGRSVEGL SRRLKRAVSE HQLLHDKGKS IQDLRRRFFL
HHLIAEIHTA EIRATSEVSP NSKPSPNTKN HPVRFGSDDE GRYLTQETNK VETYKEQPLK
TPGKKKKGKP GKRKEQEKKK RRTRSAWLDS GVTGSGLEGD HLSDTSTTSL ELDSRRH (SEQ ID
NO:2). One of skill in the art, using the N-teminual regions of either
peptide can design peptide analogs as well as fragments useful in the
methods taught herein. For example, using the first 34 amino acid
residues of PTH (MIPAKDMAKV MIVMLAICFL TKSDGKSVKK RSVS) (SEQ ID NO:3),
one of skill in the art can modify one or more amino acid residues by
insertion, deletion or substitution. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of single
or multiple amino acid residues. Insertions ordinarily will be smaller
insertions than those of amino or carboxyl terminal fusions, for example,
on the order of one to four residues. Deletions are characterized by the
removal of one or more amino acid residues from the protein sequence.
Typically, no more than about from 2 to 6 residues are deleted at any one
site within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for making
substitution mutations at predetermined sites in DNA having a known
sequence are well known, for example M13 primer mutagenesis and PCR
mutagenesis. Amino acid substitutions are typically of single residues,
but can occur at a number of different locations at once; insertions
usually will be on the order of about from 1 to 10 amino acid residues;
and deletions will range about from 1 to 30 residues. Deletions or
insertions preferably are made in adjacent pairs, i.e. a deletion of 2
residues or insertion of 2 residues. Substitutions, deletions, insertions
or any combination thereof may be combined to arrive at a final
construct. The mutations must not place the sequence out of reading frame
and preferably will not create complementary regions that could produce
secondary mRNA structure. Substitutional variants are those in which at
least one residue has been removed and a different residue inserted in
its place. Useful in the methods provided herein are PTH or PTHrP
fragments containing one or more conservative amino acid substitutions.
Also useful in the methods are peptides having 80, 90, 95, or 99%
sequence identity with the PTH/PTHrP receptor binding region of PTH or
PTHrP. Agonistic properties of the peptides can be verified by screening
for binding of the peptide to the receptor, by the ability of the peptide
to protect or repair cartilage, or by the ability to stimulate
chondrocyte proliferation and matrix synthesis.

[0049] By intermittent administration is meant a repetitive,
non-continuous administration. With regard to intermittent contact
between a peptide or small molecule and a receptor, such intermittent
exposure occurs when the peptide or molecule is not bound to the receptor
for a period of time minutes or hours or days. Thus, intermittent
administration of a peptide or small molecule to a subject means that the
peptide or small molecule has cleared sufficiently so as not to have a
physiological effect on the receptor for a sufficient period of time
before a subsequent administration.

[0050] By protecting or promoting repair is meant that degradation or
deterioration is slowed or decreased in amount or that any deterioration
or degradation is reversed in part or in full. Such protection or repair
can slow, reduce, or eliminate the clinical symptoms of the condition
being treated.

[0051] As used herein, a subject includes a mammal such as a primate
(e.g., a human), domesticated animal (such as a cat, dog, etc.),
livestock (e.g., cattle, horse, pig, sheep, goat, etc.), laboratory
animal (e.g., mouse, rabbit, rat, guinea pig, etc.) and bird. The term
does not denote a particular age or sex. Thus, adult and newborn
subjects, whether male or female, are intended to be covered. As used
herein, patient or subject may be used interchangeably and can refer to a
subject with a disease or disorder (e.g. osteoarthritis). The term
patient or subject includes human and veterinary subjects.

[0052] Disclosed are materials, compositions, and components that can be
used for, can be used in conjunction with, can be used in preparation
for, or are products of the disclosed method and compositions. These and
other materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these materials are
disclosed that, while specific reference of each various individual and
collective combinations and permutation of these compounds may not be
explicitly disclosed, each is specifically contemplated and described
herein. Similarly, various steps of a method may be recited but such
steps can be performed in each and every combination and permutation of
the steps of the regime are specifically contemplated unless specifically
indicated to the contrary. Likewise, any subset or combination of these
materials, compositions, components, or method steps is also specifically
contemplated and disclosed.

[0053] Publications cited herein and the material for which they are cited
are hereby specifically incorporated by reference in their entireties.

EXAMPLES

Example 1

Immunohistochemical Detection of Chondrocyte Maturation Markers In
Cartilage from Normal Subjects and Subjects with Mild or Severe
Osteoarthritis

[0054] Immunohistochemistry was performed on 3-4 micron sections of
paraffin embedded cartilage tissue sections from individuals diagnosed
with mild or severe osteoarthritis as well as control subjects. The
streptavidin-enzyme conjugate method was used. Appropriate fixation and
decalcification of was achieved using Immunocal (Decal Chemical Corp.;
Tallman, N.Y.). Two xylene rinses were used to remove the paraffin wax
from the tissue sections. Deparaffinization and hydration were performed
as follows: Xylene for 1-5 minutes, Xylene for 2-5 minutes, 100% Alcohol
for 1-3 minutes, 100% Alcohol for 2-3 minutes, 95% Alcohol for 1-3
minutes, 85% Alcohol for 1-3 minutes, 70% Alcohol for 1-3 minutes, and
distilled H20 for 3 minutes. In addition, tissue section microwaving
was used for antigenic recovery (antigen "unmasking"). After the tissue
sections were deparaffinized and rehydrated, endogenous peroxidase was
quenched with 3% hydrogen peroxide for 10 minutes. The slides were rinsed
in PBS and then microwaved in 0.01M citrate buffer for 1 minute at power
level 6. Afterwards, the slides remained in the hot solution for 30
minutes at room temperature. If a digestion step was necessary, slides
were digested according to standard procedures. The slides were then
drained and placed in PBS for 5 minutes. PBS was removed and the slides
were placed in a humid chamber. Normal serum was applied for 20 minutes
to saturate nonspecific binding sites (1:20 dilution).

[0055] The primary antibody dilution used was the dilution showing the
best staining with the least amount of non-specific background. The
primary antibodies were diluted in PBS/BSA and applied to the slides and
allowed to incubate at 4° C. overnight. Appropriate controls were
run for each immunohistochemical reaction and included control tissue
known to express the antigen of interest as a positive control, PBS/BSA,
and an irrelevant primary antibody.

[0056] At the beginning of day 2, the humid chamber was removed from the
refrigerator and placed at room temperature for 1 hour. Each slide was
rinsed with PBS, then placed in a rack in a staining dish with PBS for 5
minutes. The slides were removed from PBS, excess PBS was wiped away, the
slides were placed in a humid chamber, and the appropriate secondary
antibody was applied for 30 minutes at room temperature. The slides were
again rinsed in PBS in a staining dish for 5 minutes, drained and the
excess liquid was wiped away. The slides were placed in the humid chamber
and HRP Streptavidin (1:250 dilution) was applied for 30 minutes. The
slides were again rinsed in PBS in a staining dish for 5 minutes, drained
and the excess liquid wiped away, placed in the humid chamber, and then
AEC Chromagen was applied for 5-10 minutes, according to standard
procedures. The slides were drained onto an AEC absorbent pad, then
washed in distilled H2O 2 times for 5 minutes, then dipped into
hematoxylin, and rinsed in distilled H2O until water was clear. The
slides were then dipped into ammonia H2O, rinsed several times in
distilled H2O, and coverslips were added with aqueous mounting
media. The slides were covered and kept away from light to prevent fading
of the stain.

[0057] Regulatory factors known to be expressed in the growth plate were
absent in normal human adult articular cartilage but were expressed early
in cartilage degeneration (FIG. 1), including PTHrP, IHH, PTH/PTHrP
receptor, matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 13
(MMP13), Indian Hedgehog (IHH), and bone morphogenetic protein 6 (BMP6).
As shown in FIG. 1, chondrocyte maturation markers, such as PTHrP and
PTH/PTHrP receptor, were undetectable in normal cartilage but were mildly
expressed in mild osteoarthritis (low Mankin score, LM) and are strongly
expressed in moderately severe osteoarthritis (high Mankin score, HM).
The PTHrP in osteoarthritic cartilage was expressed in groups of cloning
(i.e., proliferating) chondrocytes, which is one feature of apparent
early attempts of the tissue to repair itself (FIG. 2A). The receptor,
PTHR1, was also expressed in osteoarthritic but not normal human
articular cartilage (FIGS. 1 and 2B).

Example 2

Effect of Continuous Exposure to PTHrP on Chondrocytes in Culture

[0058] PTHrP or dbcAMP stimulated smurf2 expression after 48 hr exposure
in chick chondrocytes, suggesting that chronic, continuous (as opposed to
intermittent) PTHR1 stimulation enabled escape from maturational
suppression by PTHrP and progression of the cells to hypertrophy. Without
meaning to be limited by theory, this may have been due to
down-regulation of TGF-β signaling by smurf2, allowing BMP signals
to drive hypertrophy. Thus, while continuous, longer term stimulation of
chondrocytes with PTH or PTHrP upregulated factors such as Smurf2 (FIGS.
3A-3D) that may allow escape from the proliferative to the hypertrophic
state by enhancing bone morphogenetic (BMP) signaling, intermittent
stimulation of the receptor stimulated cell proliferation and
proteoglycan synthesis without turning on the hypertrophic phenotype.

Example 3

Effect of Intermittent Exposure to PTH Fragment 1-34 on Articular
Cartilage In Vivo

[0059] Mice were anesthetized and an arthrotomy of the knee was performed.
The medial collateral ligament was surgically transected, and the
anterior horn of the medial meniscus was surgically removed to create a
mild knee injury. For the severe injury group, the anterior cruciate
ligament was also transected, causing severe joint instability in
combination with the mild meniscal and medial collateral ligament
injuries. The knee was surgically closed and animals were allowed free
movement subsequently. One group with the severe combined injuries, as
the most difficult case for cartilage protection, was treated daily with
injections of teriparatide. After sacrifice at appropriate time points,
the knee joints were removed, decalcified, embedded in paraffin, and
4-micron thick sections were stained with Alcian Blue to produce
photomicrographs as shown in FIG. 4. The resulting data show that, using
an extreme condition of mechanical insult to articular cartilage in mice
(transection of the anterior cruciate and medial collateral ligaments
plus medial menisectomy of the knee--a condition that causes severe
osteoarthritis in both mice and humans), daily injection with
teriparatide (FORTEO®; Eli Lily, Indianapolis, Ind.) dramatically
retards the development of cartilage degeneration and osteoarthritis
(FIG. 4). Teriparatide stimulated cellular repair activities with
suppression of hypertrophy or maturational changes.

Example 4

Immunohistochemical Detection of PTH/PTHrP Receptor in Meniscus Cartilage
from Normal Subjects and Subjects with Cartilage Injury

[0060] Immunohistochemistry was performed on human meniscus cartilage from
normal and injured subjects. The immunohiostochemistry was performed
generally as described above, using an antibody that specifically binds
PTH/PTHrP receptor. As shown in FIG. 5, the level of PTH/PTHrP is
upregulated in injured meniscus (right panels; 5B, 5D, 5F) as compared to
normal meniscus (left panels; 5A, 5C, and 5E). The primary antibody used
was an anti-PTH/PTHrP receptor (Upstate Cell Signaling Solutions; Lake
Placid, N.Y.; Catalog #05-517); the secondary antibody was a biotinylated
anti-mouse IgG (H+L) affinity purified made in horse (Upstate Cell
Signaling Solutions; Catalog #BA-2000); and normal horse serum was
purchased from Vector Laboratories (Burlingame, Calif.; Catalog #S-2000).

Example 5

Detection of Chondrocyte Maturation Markers in Cartilage from Mice
Following Meniscus/Ligament Injury

[0061] Expression of several chondrocyte markers was determined in mice
that had undergone meniscal injury. The expression of MMP13, ADAMTS5, and
PTHR-1 was determined and normalized to GAPDH in a control set of mice
(sham) and mice that had undergone a meniscus/ligament injury.
Chondrocyte maturation markers MMP13 and ADAMTS5 were upregulated in mice
that had undergone meniscal injury in comparison to the sham mice (FIG.
6). Further, PTHR1, the target receptor for the parathyroid hormone, was
upregulated following meniscal injury (FIG. 6).

[0062] Mice exposed to sham surgery or meniscus/ligament injury, a model
for osteoarthritis, were treated, starting immediately after surgery,
with saline, teriparatide, or PTH for 4 weeks. Daily injection of
teriparatide or PTH resulted in the upregulation of proteoglycan in the
articular cartilage as evidenced by Alcian Blue staining intensity (FIGS.
7A and 7B) demonstrating that PTH has chondroprotective effects and can
be used to stimulate PTHR1 and treat subjects with osteoarthritis.

[0063] Mice exposed to sham surgery or meniscus/ligament injury were
treated, starting immediately after surgery, with saline, teriparatide,
or PTH for 12 weeks. Daily injection of teriparatide or PTH for 12 weeks
resulted in a reduction in cartilage loss as observed by
histomorphometric measurement of joint cartilage area (FIGS. 8A and 8B).
These data show the therapeutic effect of PTH in subjects with
osteoarthritis as well as the chondroprotective effects of PTH.

[0064] Mice were allowed to develop osteoarthritis for 8 weeks following
joint surgery, as described above. After 8 weeks, the mice were treated
for 4 weeks with daily injections of teriparatide or PTH, a delayed
treatment regimen. A reduction in the loss of cartilage and regeneration
of lost cartilage was observed in mice following treatment with
teriparatide or PTH for 4 weeks as compared to the saline treated control
(FIGS. 9A and 9B). This indicates that PTHR1 receptor stimulation
regenerates lost cartilage due to osteoarthritis.

[0065] Rabbit meniscal cells were isolated from rabbit menisci and
cultured. Cultured meniscal cells were treated with PTH for 1 or 3 days.
An upregulation of mRNA for cyclin D1, a marker indicating the
stimulation of meniscal proliferation, was observed after 3 days (FIG.
10). This result is consistent with the predicted enhancement of cell
proliferation in the meniscus by the stimulation of PTHR1. Also observed
was an upregulation of mRNA for type I collagen, the predominant matrix
molecule in the meniscus (FIG. 11). This result also shows the beneficial
effect of PTHR1 stimulation on meniscal healing.

[0066] Rabbits underwent surgery to produce white zone meniscal tears. The
rabbits were treated with either saline (FIG. 12A) or teriparatide (FIG.
12B) after surgery for 4 weeks. The treated meniscus shows narrowing of
the defect with increased cellularlity and proteoglycan content (FIG.
12B), which is consistent with a stimulatory healing effect of
teriparatide on meniscal healing.

[0067] A number of methods have been described. Nevertheless, it will be
understood that various modifications may be made without departing from
the spirit and scope of the methods described herein.